Vaccine adjuvant and vaccine

ABSTRACT

The present invention includes novel polyoxyethylene/polyoxypropylene block copolymers as well as methods for making the block copolymers. The block copolymers are high molecular weight molecules and are useful as general surfactants and display enhanced biological efficacy as vaccine adjuvants.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 08/513,162, filed Aug. 9, 1995, which is a continuation-in-partof U.S. patent application Ser. No. 08/292,814, filed Aug. 9, 1994, nowabandoned.

TECHNICAL FIELD

The present invention comprises methods for synthesizing novel highmolecular weight nonionic copolymers. The present invention alsocomprises high molecular weight nonionic copolymers that are useful assurfactants and have desirable effects on living cells and organisms,including use as adjuvants in vaccines for humans and animals to augmentor otherwise modify vaccine induced immune responses.

BACKGROUND OF THE INVENTION

Nonionic block copolymers comprising blocks of polyoxypropylene andpolyoxyethylene have been synthesized and shown to have variable usesdepending the molecular size of the hydrophobic and hydrophilic regions.The commercially available nonionic copolymers are molecules that havelow molecular weight hydrophobic polyoxypropylene regions with varyingpercentages of total molecular weight hydrophilic regions attached.These nonionic copolymers are prepared by the sequential addition of twoor more alkylene oxides to a low molecular weight water-soluble organiccompound containing one or more active hydrogen atoms.

The prior art methods of synthesizing nonionic block copolymers includesthe sequential addition first of propylene oxide units and then ethyleneoxide units to a low molecular weight, water-soluble organic initiatorcompound, such as propylene glycol. The oxyalkylation steps are carriedout in the presence of an alkaline catalyst such as sodium or potassiumhydroxide. The alkaline catalyst is then neutralized and removed fromthe final product. The size of copolymers made using this technique arelimited to molecules with hydrophobic molecular weights of approximately4000, 10 to 80% of the total molecule consisting of ethylene oxide.

Other nonionic copolymers have been synthesized using nitrogencontaining molecules as the base molecule. The condensation of propyleneoxide with a nitrogen-containing reactive hydrogen compound and thesubsequent condensation of ethylene oxide therewith were carried out inthe known manner for condensing alkylene oxides with reactive hydrogencompounds. The process is normally carried out at elevated temperaturesand pressures in the presence sodium hydroxide, potassium hydroxide,sodium alkoxide, quartenary ammonium bases and the like. Thecondensation reactions can also be carried out in the presence of anacid catalyst. The manipulative steps will vary to some extent dependingupon the normal physical state of the reactive hydrogen compound.

Although nonionic block copolymers can be synthesized with low molecularweight hydrophobic regions, using conventional alkali catalyzedpolymerization methods, no one has been able to synthesize nonioniccopolymers with high molecular weight hydrophobic regions. Problems withthe synthesis of high molecular weight nonionicpolyoxyethylene/polyoxypropylene copolymers, especially with highmolecular weight hydrophobe regions, include a high degree ofunsaturation and a high degree of premature chain termination resultingin a distribution of components with low molecular weight chains alongwith the distribution of components with desirable high molecular weightchains. Using prior art methods of producingpolyoxyethylene/polyoxypropylene block copolymers results in anunacceptable variety of polymer sizes and an unacceptably high degree ofunsaturation in the polymer. This is especially undesirable when thecopolymers are to be used in biological application.

One of the needs of the medical industry is for compounds that modulatethe immune response in various ways. In addition, compounds are neededto facilitate gene transfer in cells. For example, over the past decade,the emergence of methods of gene transfer to mammalian cells hasprompted enormous interest in the development of gene-based technologiesfor the treatment of human disease. Current gene therapy technology hasfocused primarily on the use of viral and retroviral vectors whichprovide highly efficient transduction and high levels of gene expressionin vivo. The most studied are retroviral vectors, replication-defectivemurine retroviruses, which require specialized "packaging" cell linesfor their replication. Retroviral vectors integrate into chromosomes ofdividing cells leading to stable expression of the integrated gene.Also, replication-defective adenoviral and adeno-associated viralvectors have been extensively utilized. These vectors have the advantageof efficiently transducing non-dividing cells, generally do notintegrate into the host cell genome, and result in high levels oftransient gene expression. However, the use of viral methods of genetransfer for human therapy has raised safety concerns mainly due to thepotential of replication-defective viral vectors to becomereplication-competent and therefore infectious (reviewed by Mulligan,1993).

An alternative to viral gene transfer has been the use of non-viralmethods such as: cationic liposomes, delivery of ligand-DNA complexes byreceptor-mediated endocytosis, DNA coated microprojectiles and nakedDNA. Liposome-mediated gene transfer has been utilized extensively in invitro transfection studies but its application for in vivo gene deliveryhas been limited. The main disadvantage of these methods is that onlytransient gene expression is achieved and thus repeated administrationswould be necessary if continued gene expression were needed.

Recent studies have focused in the use of naked DNA for geneticimmunization. It has been shown that intramuscular inoculation of BALB/cmice with a high concentration of plasmid DNA encoding influenza Anucleoprotein results in the generation of specific CTL responses andprotection from a challenge infection of influenza A virus (Ulmer, J.B., et al. (1993) Heterologous protection against influenza by injectionof DNA encoding a viral protein. Science 259, 1745-1749). Successfulgenetic vaccination against influenza virus has also being obtained byintradermal immunization with naked DNA (Raz, E. et al., (1994)Intradermal gene immunization: The possible role of DNA uptake in theinduction of cellular immunity to viruses. PNAS 91, 9519-9523). Althoughsuccessful immunization has been achieved using DNA alone, other moreefficient methods of DNA delivery such as the use of DNA-coatedmicroprojectiles are being explored (Vahlsing, H. L., et al. (1994)Immunization with plasmid DNA using a pneumatic gun. J. Immunol. Meth.175, 11-22).

What is needed is a composition of polyoxyethylene/polyoxypropyleneblock copolymers with narrow molecular weight distribution andpolyoxypropylene hydrophobic block molecular weight higher thanapproximately 7000. Further, what is needed is a method for synthesizingnonionic polyoxyethylene/polyoxypropylene copolymers with a narrowmolecular weight distribution and high molecular weight polyoxypropylenehydrophobe. These copolymers should also have enhanced activity asadjuvants, permitting vaccination with lower amounts of antigens such asviral proteins, and display lower toxicity than conventional adjuvants.Also needed are compounds that can facilitate the transfer of genes tocells.

SUMMARY OF THE INVENTION

In accordance with the present invention, a new class ofpolyoxyethylene/polyoxypropylene copolymers, useful as surfactants andadjuvants and capable of affecting biological systems is provided. Thepresent invention provides a synthetic method and a resultingcomposition for nonionic block polyoxyethylene polyoxypropylenecopolymers with a molecular weight of the hydrophobic region that ismuch higher than block copolymers currently available. The compositionsare particularly useful as surfactants and as adjuvants in vaccines andgene therapy etc. The superior adjuvant properties of the compositionfacilitate vaccination with lower amounts of antigen.

The biologically-active copolymer of the present invention comprises ablock copolymer of polyoxyethylene (POE), which is hydrophilic, andpolyoxypropylene (POP) which is hydrophobic. The block copolymer isbuilt on a propylene glycol initiator. In a preferred embodiment of thebiologically-active copolymers of the present invention, the blockcopolymers that comprise the biologically-active copolymers of thepresent invention have the following general formulas:

    HO(C.sub.2 H.sub.4 O).sub.a (C.sub.3 H.sub.6 O).sub.b (C.sub.2 H.sub.4 O).sub.a H

wherein "b" represents a number such that the molecular weight of thepolyoxypropylene hydrophobe (C₃ H₆ O) is between approximately 7,000 and20,000 Daltons and "a" represents a number such that the percentage ofpolyoxyethylene hydrophile (C₂ H₄ O) is between approximately 1% and 40%by weight.

According to the present invention, the copolymer is synthesized usingpropylene glycol as the initiating molecule. Cesium hydroxide,monohydrate (CsOH.H₂ O) is the catalyst, usually used in a mole ratio of1:2 to 1:8 with the initiating molecule. Under reduced pressure andelevated temperatures, the propylene oxide is added by rate limitingvapor phase addition to the reaction mixture until the molecular weightof the added polyoxypropylene is at least 8000 Daltons depending uponthe size of the desired final product. Once the desired molecular weightis achieved, the addition of propylene oxide is halted. Ethylene oxideis then introduced by vapor phase addition to the reaction mixture andallowed to add to the polypropylene termini of the molecule until thepolyethylene portion of the molecule is grown to approximately 2% to 40%of the total molecular weight of the molecule. The resulting nonionicblock copolymer molecule has a high molecular weight hydrophobic region,the polyoxypropylene block, flanked by a low molecular weighthydrophilic region, the polyoxyethylene region.

Although the reaction of propylene oxide with the reactive hydrogencompound is typically carried out by simply heating a mixture of thereactants under pressure at a sufficiently high temperature, this methodis not useful as the temperatures and pressure required are excessive,control of the reaction is difficult, and the amount of low molecularweight fraction is significantly high. In addition, the materialresulting from such a method is extremely heterogeneous andpolydisperse. According to the present invention, by adding thepropylene oxide to the reaction vessel at such a rate that it reacts asrapidly as added, excess propylene oxide in the reaction vessel isavoided, which results in increased control of the reaction, and anunexpectedly improved yield of less-unsaturated and relativelyhomogeneous high molecular weight copolymer product having a highmolecular weight hydrophobic region.

The present invention includes a method of delivering therapeutic drugsto a human or animal for treating disease states such as, but notlimited to, bacterial infection and infections caused by HIV and otherDNA and RNA viruses. The present invention relates particularly tocompositions and methods for treating infectious diseases and geneticdisorders through gene therapy and intracellular delivery of antisenseoligonucleotides or other nucleic acid sequences.

The present invention also comprises use of the new copolymer as avaccine adjuvant which, when admixed with an antigen or hapten andadministered into a human or animal, will induce a more intense immuneresponse to the antigen than when the antigen is administered alone. Inmany cases, the adjuvant that is described as the present invention willincrease overall titer of antibodies specific for the vaccine antigenand induce cellular immune responses specific for the vaccine antigen.The present invention also includes vaccines comprising an antigen orgroup of antigens and the new class of polyoxyethylene/polyoxypropylenecopolymers which are present in the composition as an adjuvant.

Accordingly, it is an object of the present invention to provide acomposition and a method for making the composition comprising apolyoxyethylene/polyoxypropylene block copolymer that has an internalpolyoxypropylene block with a molecular weight of between approximately7000 and 20,000 Daltons and the polyoxypropylene block copolymer beingsubstantially free of unsaturation.

Another object of the present invention is to provide compounds that canstimulate the immune system and act as an effective vaccine adjuvant foruse in a human or animal.

Still another object of the present invention is to provide acomposition with superior adjuvant properties that facilitatesvaccination with lower amounts of antigen.

Another object of the present invention is to provide compositions thatfacilitate delivery of one or more therapeutic nucleic acid sequencefunction altering agents into the interior of a cell, such as aphagocytic cell, when admixed with a therapeutic agent.

Another object of the present invention is to provide compositions thatact synergistically with a delivered agent once inside a cell.

Still another object of the invention is to provide nonionic blockcopolymers having surfactant properties that facilitate the transmissionand introduction across cellular plasma membranes of nucleic acidsequences and compounds capable of altering nucleic acid sequencefunction.

A further object of the present invention is to provide compositions anda method for treating genetic and physiologic disorders using nucleicacid sequences and antisense oligonucleotides in combination withnonionic block copolymers.

Another object of the present invention is to provide compositions and amethod useful for manipulating the expression of genes using triplex DNAcompounds.

Yet another object of the invention is to provide DNA vaccines.

Yet another object of the present invention is to provide a method forsynthesizing polyoxyethylene/polyoxypropylene block copolymer where thepolyoxypropylene block polymer has a molecular weight of at least 7000Daltons and is substantially free of unsaturation.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a gel permeation chromatogram of CRL1005 synthesized asdescribed in Example I.

FIGS. 2a and 2b show particle size distribution of 2.5% L121 (top) and2.5% CRL1005 copolymers in phosphate buffered saline. Analysis was doneusing a Model 770 AccuSizer and test preparations of identical volume.Data are shown as frequency (total numbers)/size in μm.

FIGS. 3a to 3c show particle size distribution of 2.5% CRL1005 (top),2.5% CRL1011 (middle) and 2.5% CRL1053 copolymers in phosphate bufferedsaline. Analysis was done using a Model 770 AccuSizer and testpreparations of identical volume. Data are shown as frequency (totalnumbers)/size in μm.

FIG. 4 is a graph showing the effect of the CRL1005 from Example II inmodulating the antibody responses to a commercial influenza vaccine(FLUOGEN) in BALB/c mice as measured by the ability of antibodies inserum to inhibit the influenza virus hemagglutinin-dependentagglutination of red blood cells.

FIGS. 5a to 5c display a dose titration of FLUOGEN administered withoutadditional adjuvants. Five mice per group were immunized once with 0.5,1.5 or 4.5 μg of FLUOGEN and antibody responses examined over 60 days.Responses of individual mice are shown as the open circles and thegeometric mean for the group is shown as the solid circles. All data areshown as antibody units.

FIGS. 6a to 6f show dose titration of CRL1005 adjuvant administered withtwo dose levels of FLUOGEN. Five mice per group were immunized once with4.5 μg (upper graphs) or 1.5 μg (lower graphs) of FLUOGEN formulatedwith 5.0, 2.5 and 1.25% CRL1005 adjuvant. Antibody responses werefollowed for 60 days. Responses of individual mice are shown as the opencircles and the geometric mean for the group is shown as the solidcircles. All data are shown as antibody units.

FIGS. 7a and 7b demonstrate a summary and comparison of CRL1005 dosetitration data using two dose levels of FLUOGEN. Five mice per groupwere immunized once with 4.5 or 1.5 μg of FLUOGEN formulated with 1.25,2.5 and 5.0% CRL1005 adjuvant or without an adjuvant (0%). Antibodylevels in sera of immunized mice on day 27 are shown. Data represent thegeometric mean O 1 standard deviation for the group. All data are shownas antibody units.

FIGS. 8a and 8b show kinetics and duration of antibody responses inducedby vaccination with FLUOGEN alone or formulated with CRL1005 adjuvant.Five mice per group were immunized once with 4.5 μg of FLUOGENformulated with 2.5% CRL1005 adjuvant. Antibody responses were followedfor 6 months. Responses of individual mice are shown as the open circlesand the geometric mean for the group is shown as the solid circles. Alldata are shown as antibody units.

FIG. 9 is a graph showing the effect of CRL1005 on the antibody responseto hepatitis B surface antigen.

FIGS. 10a to 10d show serum antibody responses to ovalbumin (OVA) inC57BL6 mice following two immunizations with suboptimal doses of OVAalone or with experimental adjuvants. Six animals/group were testedusing a standard ELISA and a log₅ titration of sera (1/100-1/25,000).Data are shown as the mean absorbance (450 nm) for each serum dilutionfor individual mice.

FIGS. 11a to 11e display serum antibody responses to OVA in C57BL/6 micefollowing two immunizations with suboptimal doses of OVA formulated withclinically relevant adjuvants, alum, saponin (Quil-A, SuperfosBiosector), Ribi adjuvant (oil-in-water (O/W) containing4'-monophosphoryl lipid A, Ribi Immunochemical, Inc., Hamilton, Mont.),and CRL1005. Six animals/group were tested using a standard ELISA and alog₅ titration of sera (1/100-1/25,000). Data are shown as the meanabsorbance (450 nm) for each serum dilution for individual mice.

FIGS. 12a to 12d present OVA-specific cytotoxic T-lymphocyte (CTL)responses induced in C57BL/6 mice following two immunizations (days 0and 28) with OVA in saline (left) or formulated in aqueous solution withCRL1005 copolymer adjuvant (right). A standard ⁵¹ chromium (CR)-releaseCTL assay was used with OVA-transfected EG-7.OVA cells and control EL-4cells as the targets. Spleen cells were used as the source of precursorCTL, which were induced to mature to functional CTL effector cells, byculture for 6 days with irradiated EG-7.OVA or EL-4 cells. All testingwas done in triplicate using a titration of Effector: Target cells,shown as ratios. Data are shown as the Percent Specific Release (TestRelease--Spontaneous Release/Maximum Release--Spontaneous Release) X100.

FIGS. 13a to 13f show serum antibody responses to OVA in C57BLJ6 micefollowing two immunizations with OVA administered s.c. orally or nasallywithout CRL1005 (upper) or with CRL1005 (lower). Sera from five animalsper group were evaluated using a standard ELISA and a log₅ titration ofsera (1/100-1/12,500). All testing was done in duplicate. Data are shownas the mean absorbance (450 nm) for each serum dilution for individualmice (•--•). The background for the ELISA was established by omission ofthe mouse serum(o--o).

FIG. 14 shows the initial screening of poloxamers using a transfectionmethod described for liposome-mediated DNA transfection.

FIG. 15 shows transfection using poloxamers 1012, 1029 and 1030.

FIG. 16 shows the effect of temperature on transfection efficiency.

FIG. 17 shows the variability between poloxamer concentration andtransfection efficiency.

FIG. 18 shows the effect of mixing the poloxamer-DNA mixtures byvortexing or emulsification with a syringe.

FIG. 19 shows the effect of rotating the plates for 24 hours during thetransfection.

FIG. 20 shows the effect scaling up the assay conditions on efficiencyof transfection.

FIG. 21 shows the antibody response to genetic vaccination with pATCgDplasmid DNA.

FIG. 22 shows the antibody response to genetic vaccination with pATCgDplasmid DNA prior to ocular infection with HSV-1.

FIG. 23 shows genetic vaccination against ocular HSV-1 in BALB/c mice.

FIG. 24 shows genetic vaccination against HSV-1 skin infection in SKH-1(Hairless) mice.

FIG. 25 shows the severity of HSV infection in SKH-1 (Hairless) miceafter genetic immunization.

DETAILED DESCRIPTION OF THE INVENTION

The term "antigen" is defined as anything that can serve as a target foran immune response. The term "adjuvant" means compounds that, when usedin combination with specific vaccine immunogens in formulations, augmentor otherwise alter or modify the resultant immune responses. The term"vaccine" is defined herein as a suspension or solution of antigenicmoieties, usually consisting of inactivated infectious agents, or somepart of the infectious agents, that is injected into the body to produceactive immunity. The antigenic moiety making up the vaccine can beeither a live or killed microorganism, or a natural product purifiedfrom a microorganism or other cell including, but not limited to tumorcells, a synthetic product, a genetically engineered protein, peptide,polysaccharide or similar product or an allergen. The antigenic moietycan also be a subunit of a protein, peptide, polysaccharide or similarproduct. The term "cell mediated immunity" is defined as an immuneresponse mediated by cells or the products they produce, such ascytokines, rather than by antibody. It includes, but is not limited to,delayed type hypersensitivity and cytotoxic T cells. The term "adjuvant"as used herein is any substance whose admixture with an injectedimmunogen modifies the immune response. Modification of the immuneresponse means augmentation, intensification, or broadening thespecificity of either or both antibody and cellular immune responses.Modification of the immune response can also mean decreasing orsuppressing certain antigen-specific immune responses such as theinduction of tolerance. A "hapten" is defined herein as a substance thatreacts selectively with appropriate antibodies or T cells but the haptenitself is usually not immunogenic. Most haptens are small molecules orsmall parts of large molecules, but some macromolecules can alsofunction as haptens.

The present invention comprises a method of synthesizing high molecularweight polyoxyethylene/polyoxypropylene block copolymers. The presentinvention also includes high molecular weightpolyoxyethylene/polyoxypropylene block copolymers. The nonionic blockcopolymers have the following general formula:

    HO(C.sub.2 H.sub.4 O).sub.a (C.sub.3 H.sub.6 O).sub.b (C.sub.2 H.sub.4 O).sub.a H

wherein b represents a number such that the molecular weight of thehydrophobe (C₃ H₆ O) is between approximately 7000 and 20,000 Daltonsand a represents a number such that the percentage of hydrophile (C₂ H₄O) is between approximately 1% and 40% by weight.

According to the present invention, the nonionic block copolymers aresynthesized using the following procedure. An initiator molecule such aspropylene glycol, ethylene glycol or diethylene glycol, is mixed withthe catalyst, cesium hydroxide, monohydrate in mole ratios fromapproximately 2:1 to 8:1. Throughout the synthesis, all reactants andreactions are kept in an oxygen-free environment.

The catalyst and the initiator are placed in a glass-lined pressurereactor and heated. Propylene oxide gas is added to the reactor vesselby a rate limiting, vapor phase addition method. The reactants in thereactor are stirred and heated, at temperatures ranging from 90° C. to120° C. The propylene oxide-initiator molecule reaction is allowed tocontinue until the product polymers are the desired molecular weight, asdetermined, for example, by gel permeation chromatography. The molecularweights of the hydrophobic portion of the molecule (C₃ H₆ O) can rangefrom 7,000 to 20,000 Daltons, more particularly from 9,000 to 15,000Daltons, and even more particularly, from 10,000 to 14,000 Daltons. Thesize of the hydrophobic portion of the molecule can be varied bychanging the amount of propylene oxide used in the reaction. Afterreacting the propylene oxide required, ethylene oxide is added to thepolypropylene oxide hydrophobe. The polyoxyethylene portion of themolecule is preferably between 1% and 30% of the total weight of thepolymer with a more preferable range of between 3% and 25% of the totalweight of the polymer.

The amount of ethylene oxide (EtO) to be used in the next part of thesynthesis is determined from the amount of propylene oxide (PrO) used.For example, the amount of ethylene oxide required to produce a polymerwith a total polyethyleneoxide content of 5% is calculated by thefollowing formula: ##EQU1##

The grams of ethylene oxide required equals the weight of propyleneoxide over 19. The ethylene oxide is then added to the reactor vesselunder the same conditions as above. After the ethylene oxide is reacted,the molecular weight of the polymer is determined using gel permeationchromatography.

The product polymer is then preferably treated with magnesium silicate(Magnesol), diatomaceous earth (Celite), and water. The water,diatomaceous earth, and magnesium silicate are added in at least threealiquots to the reactor which is maintained in the oxygen-freeenvironment, at a high heat with stirring, over six hours. It has beenfound that adding these reagents in aliquots more effectively removesthe residual catalyst than adding all at once. At the end of theseadditions, the reactor vessel is allowed to return to room temperature.Again, samples of the product polymer are taken for molecular weightdetermination by gel permeation chromatography. While maintaining theoxygen-free environment, the product polymer is filtered and packaged.

The high molecular weight nonionic copolymers of the present inventionare useful as general surfactants and as adjuvants in vaccines. Vaccinesmade with the high molecular weight copolymers induce higher antibodytiters in animals than do vaccines which do not contain the copolymers(see Examples III and IV below). Furthermore, use of the composition ofthe present invention enables effective vaccination with lower amountsof antigen in the vaccine. The antigen component of the vaccine maycomprise one or several antigenic molecules such as haptens, proteins,nucleic acids, tumor cells and antigens from various sources such asinfectious agents.

An effective vaccine must induce an appropriate response to the correctantigen or antigens. There are several distinct types of immuneresponses which vary in their ability to confer protection againstparticular diseases. For example, antibodies may confer protectionagainst bacterial infections, but cell mediated immunity is required foreliminating from the body many viral infections and tumors. There aremultiple distinct types of antibody and cell-mediated immune responses.Cell-mediated responses are divided into two basic groups: 1)delayed-type hypersensitivity in which T cells act as helper orsuppressor cells indirectly via macrophages and other cells or cellproducts and via indirect interactions through products secreted fromthe T cells such as cytokines, and 2) cytotoxicity in which specializedT-cells specifically and directly attack and kill infected cells.

Thus, the present invention comprises an improved adjuvant. In oneembodiment of the present invention, an antigen is admixed with aneffective amount of an adjuvant, the adjuvant comprises a surface-activecopolymer having the following general formula:

    HO(C.sub.2 H.sub.4 O).sub.a (C.sub.3 H.sub.6 O).sub.b (C.sub.2 H.sub.4 O).sub.a H

wherein "b" represents a number such that the molecular weight of thehydrophobe (C₃ H₆ O) is between approximately 7000 and 20,000 Daltonsand "a" represents a number such that the percentage of hydrophile (C₂H₄ O) is between approximately 1% and 40% by weight.

A preferred surface-active copolymer that can be used as a vaccineadjuvant has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.a (C.sub.3 H.sub.6 O).sub.b (C.sub.2 H.sub.4 O).sub.a H

wherein "b" represents a number such that the molecular weight of thehydrophobe (C₃ H₆ O) is between approximately 9000 Daltons and 15,000Daltons and "a" represents a number such that the percentage ofhydrophile (C₂ H₄ O) is between approximately 3% and 35%.

Another preferred surface-active copolymer that can be used as a vaccineadjuvant has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.a (C.sub.3 H.sub.6 O).sub.b (C.sub.2 H.sub.4 O).sub.a H

wherein "b" represents a number such that the molecular weight of thehydrophobe (C₃ H₆ O) is approximately 9000 Daltons and "a" represents anumber such that the percentage of hydrophile (C₂ H₄ O) is approximately3%.

Another preferred surface-active copolymer that can be used as a vaccineadjuvant has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.a (C.sub.3 H.sub.6 O).sub.b (C.sub.2 H.sub.4 O).sub.a H

wherein "b" represents a number such that the molecular weight of thehydrophobe (C₃ H₆ O) is approximately 11000 Daltons and "a" represents anumber such that the percentage of hydrophile (C₂ H₄ O) is approximately5%.

Antigens that can be used in the present invention are compounds which,when introduced into a mammal, will result in the formation ofantibodies and cell mediated immunity. Representative of the antigensthat can be used according to the present invention include, but are notlimited to, natural, recombinant or synthetic products derived fromviruses, bacteria, fungi, parasites and other infectious agents inaddition to autoimmune diseases, hormones or tumor antigens which mightbe used in prophylactic or therapeutic vaccines and allergens. The viralor bacterial products can be components which the organism produced byenzymatic cleavage or can be components of the organism that wereproduced by recombinant DNA techniques that are well-known to those ofordinary skill in the art. The following is a partial list ofrepresentative antigens:

Viruses

Rotavirus

Foot and mouth disease

Influenza

Parainfluenza

Herpes species,

Herpes simplex,

Epstein Barr virus

Chicken pox,

pseudorabies

Cytomegalovirus

Rabies

Polio

Hepatitis A

Hepatitis B

Hepatitis C

Hepatitis E

Measles

Distemper

Venezuelan equine encephalomyelitis

Feline leukemia virus

Reovirus

Respiratory sycytial virus

Lassa fever virus

Polyoma tumor virus

Canine parvovirus

Papilloma virus

Tick borne encephalitis

Rinderpest

Human rhinovirus species

Enterovirus species, Mengo virus

Paramyxovirus

Avian infectious bronchitis virus

HTLV 1

HIV-1

HIV-2

Influenza A and B

LCMV (lymphocytic choriomeningitis virus)

Parovirus

Adenovirus

Togavirus (rubella, yellow fever, dengue fever)

Bovine respiratory syncicial virus

Corona virus

Bacteria

Bordetella pertussis

Brucella abortis

Escherichia coli

Salmonella species, salmonella typhi

Streptococci

Vibrio (V. cholera, V. parahaemolyticus)

Shigella

Pseudomonas

Brucella species

Mycobacteria species (tuberculosis, avium, BCG, leprosy,)

Pneumococci

Staphlylococci

Enterobacter species

Rochalimaia henselae

Pasterurella (P. haemolytica, P. multocida)

Chlamydia (C. trachomatis, C. psittaci, Lymphogranuloma venereum)

Syphilis (Treponema pallidum)

Haemophilus species

Mycoplasmosis

Lyme disease (Borrelia burgdorferi)

Legionnaires' disease

Botulism (Colstridium botulinum)

Corynebacterium diphtheriae

Yersinia entercolitica

Ricketsial Infections

Rocky mountain spotted fever

Thyphus

Ehrlichia

Parasites and Protozoa

Malaria (Plasmodium. falciparum, P. vivax, P. malariae)

Schistosomes

Trypanosomes

Leishmania

Filarial nematodes

Trichomoniasis

Sarcosporidiasis

Taenia (T. saginata, T. solium)

Leishmania

Toxoplasma gondii

Trichinelosis (Trichinella spiralis)

Coccidiosis (Eimeria species)

Fungus

Cryptococcus neoformans

Candida albicans

Apergillus fumigatus

Coccidioidomycosis

Subunit recombinant proteins

Herpes simplex

Epstein Barr virus

Hepatitis B

Pseudorabies

Flavivirus, Denge, Yellow fever

Neisseria gonorrhoeae

Malaria: circumsporozoite protein, merozoite protein

Trypanosome surface antigen protein

Pertussis

Alphaviruses

Adenovirus

Proteins

Diphtheria toxoid

Tetanus toxoid

Meningococcal outer membrane protein (OMP)

Streptococcal M protein

Hepatitis B

Influenza hemagglutinin

Cancer antigen, tumor antigens

Toxins, Exotoxins, Neurotoxins

Cytokines and Cytokine receptors

Monokines and monokine receptors

Synthetic peptide

Malaria

Influenza

Foot and mouth disease virus

Hepatitis B, Hepatitis C

Polysaccharide

Pneumococcal polysaccharide

Haemophilis influenza

polyribosyl-ribitolphosphate (PRP)

Neisseria meningitides

Pseudomonas aeruginosa

Klebsiella pneumoniae

Oligosaccharide

Pneumococcal

Allergens

Plant pollens

Animal dander

dust mites

Haptens are compounds which, when bound to an immunogenic carrier andintroduced into a chordate, will elicit formation of antibodies specificfor the hapten. Representative of the haptens are steroids such asestrogens and cortisones, low molecular weight peptides, other lowmolecular weight biological compounds, drugs such as antibiotics andchemotherapeutic compounds, industrial pollutants, flavoring agents,food additives, and food contaminants, and/or their metabolites orderivatives.

When used as an adjuvant, the polyoxyethylene/polyoxypropylene blockcopolymer of the present invention can be administered to a human oranimal by a variety of routes including, but not limited to,intramuscular injection, intravenous injection, intraperitonealinjection, orally, rectal, vaginal, sublingually, and nasally.

The present invention also comprises a therapeutic delivery compositioneffective for treating a disease state comprising an administerableadmixture of an effective amount of a therapeutic compound capable ofaltering nucleic acid sequence function and an effective amount of asurface active nonionic block copolymer having the following generalformula:

    HO(C.sub.2 H.sub.4 O).sub.a (C.sub.3 H.sub.6 O).sub.b (C.sub.2 H.sub.4 O).sub.a H

wherein "b" represents a number such that the molecular weight of thehydrophobe (C₃ H₆ O) is between approximately 7000 and 20,000 Daltonsand "a" represents a number such that the percentage of hydrophile (C₂H₄ O) is between approximately 2% and 40% by weight.

Another preferred surface-active copolymer that can be used as atherapeutic delivery agent has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.a (C.sub.3 H.sub.6 O).sub.b (C.sub.2 H.sub.4 O).sub.a H

wherein "b" represents a number such that the molecular weight of thehydrophobe (C₃ H₆ O) is approximately 13000 Daltons and "a" represents anumber such that the percentage of hydrophile (C₂ H₄ O) is approximately5%.

Another preferred surface-active copolymer that can be used as atherapeutic delivery agent has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.a (C.sub.3 H.sub.6 O).sub.b (C.sub.2 H.sub.4 O).sub.a H

wherein "b" represents a number such that the molecular weight of thehydrophobe (C₃ H₆ O) is approximately 11000 Daltons and "a" represents anumber such that the percentage of hydrophile (C₂ H₄ O) is approximately15%.

A particularly useful composition is an admixture of a compound capableof altering gene expression and/or protein translation, such as anantisense oligonucleotide, a triplex DNA compound, a ribozyme or othercompound capable of altering nucleic acid sequence function, and theabove-described nonionic block copolymer.

The composition of the present invention can be administered by a numberof routes including, but not limited topical, transdermal, oral,trans-mucosal, subcutaneous injection, intravenous injection,intraperitoneal injection and intramuscular injection.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE I

Synthesis of CRL1005

The poloxamer, CRL1005, is a tri-block copolymer of polyoxyethylene(POE) and polyoxypropylene (POP) with the middle POP hydrophobe havingan average molecular weight of approximately 12000 Daltons, and thepolymer having 5% POE. The initiator for the synthesis is propyleneglycol. The catalyst is cesium hydroxide monohydrate (CsOH.H₂ O) in amole ratio of 2:1 (initiator:catalyst).

About 3.52 gm of cesium hydroxide was dispensed and then transferredinto a glass-lined pressure reactor. The reactor was heated under vacuumat temperature of 100° C. for approximately 20 hours to dry the CsOH.After the reactor has cooled to a temperature below 50° C., an amount of3.18 gm of the initiator, propylene glycol, was weighed and charged intothe reactor.

A reservoir containing propylene oxide (PrO) was connected to thereactor. The reservoir was maintained at 30° C. in a silicone oil heatbath. Only the propylene oxide vapors from the reservoir were allowed toreact with the propylene glycol initiator in the reactor. The reactorwas heated and maintained at 100° C. while stirring throughout thepropylene oxide addition procedure. The PrO addition reaction wascontinued until a total of 1073.72 gm of PrO was added to the reactor.

Samples were taken for analysis for molecular weight determination,using polyethylene glycol (PEG) standards to establish the calibrationcurve.

56.85 gm of ethylene oxide was charged to the reactor under the samecondition as the PrO, except the ethylene oxide glass reservoir was keptat room temperature. Ethylene oxide in the reactor was allowed tocontinue to react to completion. At this point, more samples were takenfor gel permeation chromatography (GPC) analysis.

The polymer was treated with magnesium silicate (Magnesol)(approximately 4×wt. of CsOH.H₂ O), diatomaceous earth (Celite)(approximately 0.3×wt of Magnesol), and water (approximately 0.11×wt. ofCsOH.H₂ O). An amount of 0.37 gm of water was dispensed and stirred intothe reactor under a slight purge of nitrogen. A mixture of 14.15 gm ofMagnesol and 4.32 gm of Celite was separated into three nominally equalportions. Each portion of the mixture was added, in three 2 hourintervals, to the reactor while heating at 110° C. and stirring atapproximately 60 rpm under a slight nitrogen purge. Adding the mixturein several portions is important to effectively remove the residualcatalyst. At the end of the 6 hour treatment period, the reactor wasallowed to cool to room temperature before in-process samples were takenfor GPC analysis.

The treated polymer was filtered. The polymer and filter funnel wereheated to 70° C. Nitrogen was applied through a pressure holder toapproximately 40 psig to facilitate the filtration process. The polymerwas collected in glass Quorpak bottles for storage under nitrogen andrefrigeration.

EXAMPLE II

Physical Properties of the CRL1005

The CRL1005 synthesized in Example I was analyzed using gel permeationchromatography and nuclear magnetic resonance. The results of theanalysis are as follows:

    ______________________________________                                        GPC ANALYSIS                                                                  ______________________________________                                        Peak Mwt            13763                                                     Wt. Av Mwt          11621                                                     % of Low Mwt        20.64                                                     Unsaturation, meq/gm                                                                              0.0561                                                    Percent EO, by NMR  5.70                                                      ______________________________________                                         where Mwt is molecular weight                                            

The fractions were characterized by gel permeation chromatography usingpolyethylene glycol (PEG) standards, NMR, and unsaturation. Gelpermeation chromatography provided different average molecular weights.Percentage of ethylene oxide units was determined from NMR. Unsaturationwas measured by wet chemistry and provided amount of --CH═CH-- groupspresent in the end groups. FIG. 1 shows a gel permeation chromatogram ofCRL1005.

EXAMPLE III

Particle Formation by Block Copolymers

The nonionic block copolymers that have been previously evaluated asvaccine adjuvants, such as L121 and L141, are not soluble in aqueousbuffers. When mixed in aqueous solutions the individual polymermolecules bind to each other to form strands and ultimately anunorganized matrix or gel-like material is formed. As such, thesecopolymers have been used exclusively in emulsions where copolymermolecules bind to the oil/water interface. The copolymers with the largehydrophobic POP components, such as CRL1005, form small uniformed sized(1-2 μm) particles in aqueous buffers (FIG. 2).

The amount of the hydrophilic POE component also effects the formationof particles in this size range. Increasing the POE content from 5% to10% reduces the number of particles formed by 90% and higher amounts ofPOE essentially eliminate particle formation. In the example shown, theCRL1005 with a molecular weight of 12,000 and 5% POE forms 1-2 μmparticles whereas the CRL1011 with a molecular weight of 11,200 and 10%POE is 90% less efficient at forming particles (FIG. 3). The CRL1053with a molecular weight of 13,200 and 40% POE cannot form 1-2 μmparticles.

Particulate vaccine antigens are generally more immunogenic wheninjected into animals than are soluble antigens. These properties havebeen demonstrated using several types of particulate vaccineadjuvant/delivery systems, including liposomes and poly-lactideparticles. The properties of these systems that support their use inVaccines include (1) production using biodegradable or otherwisenontoxic materials, (2) ability to produce small particles, 1-10 μmrange, and (3) the ability to encapsulate or bind to vaccine antigens.These are properties that are shared by the large, adjuvant activecopolymers, such as CRL1005.

Particulate vaccine antigens can also be delivered orally. Particulateantigens are more effectively `taken-up` by Peyer's patches andtherefore are more efficient at inducing mucosal immune responses. Theparticulate property in itself appears to increase the ability of theimmunogen to gain access to the Peyer's patches with the optimum sizerange being 1-10 μm. Again, these are properties that are shared by thelarge, adjuvant active copolymers.

EXAMPLE IV

The effect of the CRL1005 from Example II in modulating the serologicalresponses to a commercial influenza vaccine was examined in Balb/c mice.The commercial vaccine employed was obtained from Parke-Davis (FLUOGEN®)and was the vaccine available during the 1993 influenza season. Thecommercial vaccine consists of hemagglutinin (HA) and other viralcomponents from A/TEXAS/36/91 (H1N1), A/BEIJING/32/92 (H3N2), andB/PANAMA/45/90 and contains 15 μg of each HA antigen. For experimentalevaluation, the commercial vaccine was mixed in equal proportions witheither buffer (0.9% NaCl;) or of copolymer (5% CRL1005, 0.9% NaCl). Micewere injected with 100 μl of the two vaccines. Serum samples wereobtained prior to injection, on day 14, day 28, and day 70.

Two serological assays were employed to measure anti-influenza specificantibodies in the sera of individual immunized mice. One is anenzyme-linked immunosorbent assay (ELISA) and the other an assay tomeasure the ability of antibodies in serum to inhibit the influenzavirus hemagglutinin-dependent agglutination of red blood cells. In thecase of the latter assay, the levels of antibodies are directlycorrelated with the ability to neutralize virus, whereas the ELISA assaydetects antibodies which may or may not be functional. Thus, the twoassays independently measure serological responsiveness to thehemagglutinin (HA).

Hemagglutination Inhibition

Functional antibody capable of neutralizing the influenza hemagglutininwas assayed by hemagglutination inhibition (HAI), essentially asdescribed by the CDC Manual (Concepts and Procedures for LaboratoryBased Influenza Surveillance, 1982. U.S. Department of Health and HumanServices/Public Health Service/Centers for Disease Control). Serialdilutions of test sera were examined for their ability to neutralize thehemagglutinating activity of 8 units of purified hemagglutinin onchicken red blood cells. Purified hemagglutinins from the following 3strains were used: A/YAMAGATA/32/89 (HINI), A/KISHU/54/89 (H3N2),B/AICHI/54/89. Titers are expressed as the highest serum dilution givingcomplete neutralization of the hemagglutinin. (FIG. 4)

EXAMPLE V

Dose Escalation and Kinetic Studies of Copolymer Adjuvant CRL1005 in aCommercial Influenza Virus Vaccine

The effects of supplementing the Parke-Davis influenza virus vaccine(FLUOGEN) with copolymer adjuvant CRL1005 were investigated. Theantibody titers and kinetics of antibody responses were examined as theendpoints.

A. Experimental Design

The design and results of three separate experiments are presented.

Experiment 1: Dose titration studies of the influenza virus vaccine weredone to identify doses that induced measurable antibody responses aftera single immunization in BALB/c mice. Antibody responses were followedfor 60 days and dose levels that were optimally and suboptimallyimmunogenic were identified.

Experiment 2: Vaccine formulations containing these dose levels andsupplemented with increasing amounts of adjuvant-active CRL1005 polymerwere evaluated. Again, antibody responses were followed for 60 days andadjuvant-active doses of CRL1005 adjuvant were identified.

Experiment 3: Kinetics and duration of antibody responses were evaluatedby extension of the observation and antibody testing period to 6 months.

Parke-Davis FLUOGEN influenza virus vaccine, from the 1993-1994 season,was used as both the vaccine and as the source of viral proteins formeasuring antibodies. BALB/c mice were used, five per group, in groupsorganized based on vaccine formulations. The adjuvant is a nonionicblock polymer designated CRL1005. An aqueous formulation was used forthese studies. This formulation consisted of the appropriateconcentration of CRL1005 polymer and FLUOGEN® mixed in saline. Under theformulation conditions used, the polymer forms 1-2 μm particles with theimmunogen. The aqueous formulations were used without additionalpreparation. Vaccine formulations were administered by subcutaneousneedle injection, total volume of 100 μl.

Ninety mice (C57BL/6) were divided into 9 test and control groups of 10mice per group. The mice were immunized twice with 28 days betweenimmunizations. Vaccines were administered by needle injectionsubcutaneously (s.c.) in a total volume of 200 μl/dose. The groups ofmice were immunized according to the following format: Group 1--vehiclecontrol (phosphate buffered saline-PBS); Group 2--15 μg of the OVA inPBS; Group 3--15 μg of the OVA adsorbed to 125 μg of alum; Group 4--15μg of the OVA formulated with CFA in a water-in-oil (W/O) emulsion,second immunization was given with IFA; Group 5--15 μg of the OVAformulated with 20 μg Quil-A; Group 6--15 μg of OVA formulated in a W/Oemulsion containing CRL1005, 2.5% concentration of 5 mg/200 μl dose;Group 7--15 μg of OVA formulated with Ribi adjuvant as an oil-in water(O/W) emulsion; Group 8--15 μg of the OVA formulated with CRL1005, 2.5%final concentration or 5 mg/200 μl dose; Group 9--15 μg of the OVAformulated with CRL1005, 5.0% final concentration or 10 mg/200 μl dose.

B. Measurement of Vaccine-Induced Antibody Responses

Blood was collected via the retro-orbital sinus at various time-points,usually at 2 and 4 week intervals. Antibody levels were determined usinga standard ELISA and the FLUOGEN vaccine as the source of viralproteins. A standard positive control antiserum was prepared in BALB/cmice using the FLUOGEN vaccine formulated with emulsion-based adjuvant,to induce very high-titered antibody responses in the serum. Thisantiserum was used to validate the ELISA and to establish a standardcurve to permit direct comparison of data obtained from experimentsperformed at different times. This antiserum was defined arbitrarily ascontaining 10,000 antibody binding Units and was used as the standard todetermine antibody Units for all samples obtained from experimentallyvaccinated mice.

C. Results

1. Identification of immunogenic doses of FLUOGEN

Antibody responses induced following a single immunization with 4.5, 1.5or 0.5 μg of FLUOGEN are shown in FIG. 5. All doses were immunogenic butthe 4.5 μg dose induced significantly higher antibody responses than the1.5 and 0.5 μg doses and it was selected as the optimal dose level.Since the responses induced by the two lower concentrations were notsignificantly different, the 1.5 μg dose was selected as the suboptimaldose level for further studies.

2. Effect of Optivax adjuvant on antibody levels, kinetics and duration

The adjuvant effects of three different concentrations, 5.0, 2.5 and1.25%, of the CRL1005 polymer are shown in FIG. 6. All concentrationswere active as adjuvants. Increases in antibody titers induced using the4.5 μg dose of FLUOGEN ranged from 2-8 fold, with the greatest increasesseen on day 27. The highest concentration of CRL1005, 5.0%, resulted inthe greatest augmentation of the antibody responses for the 4.5 μg doseof FLUOGEN. Augmentation of antibody responses was more pronounced usingthe 1.5 μg dose of FLUOGEN. In these animals, CRL1005 increased antibodylevels more than 10 fold and again the most significant differences wereobserved on day 27. Antibody levels were increased to levels that werenot significantly different from those obtained using the higher dose ofFLUOGEN (FIG. 7). These data suggest that the CRL1005 polymer adjuvantmay facilitate the use of lower levels of viral proteins in vaccines.

The kinetics of the primary antibody responses were shown in FIGS. 5 and6, but these data were obtained through a period of 60 days. To evaluatethe effect of CRL1005 polymer adjuvant on antibody duration, mice wereimmunized a single time with either 4.5 μg of FLUOGEN alone or with thesame formulation supplemented with 2.5% CRL1005 and antibodies weremeasured within the first 60 days and again at 6 months. The results areshown in FIG. 8. Antibody levels continued to increase throughout the 6month study period. These data indicate that the CRL1005 polymeradjuvant may prove useful for inducing long-term protection using only alimited number of vaccinations, preferably a single vaccination.

The potential value of adjuvants as components in influenza vaccines hastherapeutically and biologically significant implications particularlyfor augmentation of immune responses in the elderly. This studydemonstrated the potential utility of the CRL1005 polymer adjuvant as acomponent of an experimental influenza virus vaccine based on thecommercial FLUOGEN vaccine. The results showed the adjuvant activity ofthe CRL1005 polymer adjuvant in a simple aqueous formulation withFLUOGEN. Adjuvant formulations induced higher antibody titers whichcontinued to increase with time. The CRL1005 polymer adjuvant augmentedantibody responses to suboptimal dose levels of the FLUOGEN to an evengreater extent. The antibody titers reached levels similar to thoseinduced using the higher FLUOGEN dose suggesting that lower amounts ofvaccine immunogen may be used in adjuvanted formulations. Thus, theaddition of CRL1005 adjuvant to vaccine formulations may increaseimmunogenicity and allow for reduction in the amounts of proteinimmunogen(s) that are required.

EXAMPLE VI

Improved Activity of Vaccines

The influence of formulations containing the CRL1005 of Example II onthe antibody response to hepatitis B surface antigen was compared with avaccine containing aluminum hydroxide, a vaccine containing CRL1005 andaluminum hydroxide, and a vaccine containing no additions. The vaccineswere made by admixing a recombinant hepatitis B surface antigen (HBsAg)with each of the additional vaccine components so that the final HBsAgconcentration in each vaccine was 5 μg/ml and:

a. 2.5% CRL1005, 0.9% NaCl

b. 0.25 mg/ml Al(OH)₃, 0.9% NaCl

C. 0.9% NaCl

Groups of 6 or 8 BALB/c mice each received a single 100 μl dose of theabove vaccines subcutaneously, so that each animal was injected with 0.5μg of HBsAg. Serum samples were obtained from each individual animalprior to vaccination, on day 15 and on day 28.

Murine antibody concentrations against HBsAg were measured with thecommercially available AUSAB®EIA (Abbott Laboratories, Abbott Park,Ill.) which uses the bridging principle to detect antibody. Specificantibody in the sample initially binds with one binding site to HBsAgimmobilized on a polystyrene bead and secondarily binds to Biotinconjugated HBsAg with the other to create an antigen-antibody sandwich.The assay is therefore not species specific and can be utilized tomeasure murine as well as human anti-HBsAg. Murine antibody was assayedat a 1:10 dilution or higher and quantitated against a human serumstandard curve. Data were expressed as milli international units (mIU)per ml, calculated by multiplying the assay value by a factor of 10.

Results of the immunization are presented in FIG. 9. The quantity ofHBsAg-specific antibodies detected in individual mouse sera is expressedas geometric mean titer. The animals that received a single injection of0.5 μg HBsAg and 2.5 mg of CRL1005 had at least 4 times as much antibodyagainst the HBsAg as did animals which received 0.5 μg of HBsAg in alum.The amount of alum which was included in the two formulations wasequivalent to the accepted amount of alum in currently licensed andmarketed recombinant hepatitis B vaccines. Also, the vaccine containingno CRL1005 was only marginally immunogenic, inducing barely detectableanti-HBsAg responses. None of the animals which were vaccinated with the0.5 μg HBsAg in saline showed measurable antibody responses by day 28,as determined by those individuals having greater than 100 mIU/ml (Table1). Because sera were diluted 1:10 for assay, a value less than 100 wasconsidered below the cutoff for significance. A serum level of 10 mIU/mlis considered protective in humans, usually obtained on undiluted serum.50% of the animals that received a single injection of HBsAg withCRL1005 induced measurable antibody responses to greater than 100mIU/ml, whereas in only 25% of the animals were measurable antibodyresponses induced to the vaccine formulated with alum alone.

                  TABLE I                                                         ______________________________________                                        Seroconversion rates 28 days following                                        a single injection of 0.5 μg HBsAg                                                    a-HBsAG    Seroconversion                                                                           Increase over                                Vaccine    GMT        rate (percent)                                                                           control                                      ______________________________________                                        2.5% CRL1005                                                                             127        3/6 (50)   4                                            0.25 mg alum                                                                              31         2/8 (25%) 1                                            (control)                                                                     No additions                                                                              2         0/8 (0)      0.06                                       ______________________________________                                    

EXAMPLE VII

Evaluation of Antigen-Specific Antibody and Cytokine Responses InducedUsing Different Adjuvants and Suboptimal Dose of Ovalbumin

A. Test Reagents, Animal Requirements and Experimental Design

The immune responses induced by experimental subunit vaccineformulations based on a prototype immunogen combined with differentadjuvants including CRL1005 were evaluated. Ovalbumin (OVA) was selectedas the vaccine immunogen since dose levels of OVA for obtaining optimaland suboptimal immune responses had been established previously. Inaddition, OVA was selected because of the availability of a model toassess cytotoxic T-lymphocyte responses in C57BL/6 mice, based on theEG-7 OVA cell line (American Type Culture Collection, Rockville, Md.)(See Moore, et al., "Introduction of soluble protein into the class Ipathway of antigen processing and presentation" Cell 54:777 (1988)) thatis stably transformed with OVA and expresses OVA peptides associatedwith class I MHC antigens. OVA was administered at a dose of 15 ug/dosein a total volume of 100 ul. This dose of OVA had been established asnon-immunogenic unless combined with an adjuvant.

Ninety mice (C57BL/6) were divided into 9 test and control groups of 10mice per group. The mice were immunized twice with 28 days betweenimmunizations. Vaccines were administered by needle injectionsubcutaneously (s.c.) in a total volume of 100 μl/dose. The groups ofmice were immunized according to the following format: Group 1--vehiclecontrol (phosphate buffered saline-PBS); Group 2--15 μg of the OVA inPBS; Group 3--15 μg of the OVA adsorbed to 125 μg of alum; Group 4--15μg of the OVA formulated with CFA in a water-in-oil (W/O) emulsion,second immunization was given with incomplete Freund's aduvant; Group5--15 μg of the OVA formulated with 20 μg Quil-A; Group 6--15 μg of OVAformulated in a W/O emulsion containing CRL1005, 2.5% concentration of 5mg/200 μl dose; Group 7--15 μg of OVA formulated with Ribi adjuvant asan oil-in water (O/W) emulsion; Group 8--15 μg of the OVA formulatedwith CRL1005, 2.5% final concentration or 5 mg/200 μl dose; Group 9--15μg of the OVA formulated with CRL1005, 5.0% final concentration or 10mg/200 μl dose.

B. Measurement of Antigen-Specific Immune Responses

To evaluate antibody responses, 4 weeks after the second immunizationblood was collected via the retro-orbital sinus from 6 mice/group andsera was recovered for serological assay.

To evaluate cytokine responses, spleens were removed aseptically fromthe remaining 4 mice in each group and mononuclear cells recovered.Splenic mononuclear cells were cultured at a concentration of 4×10⁶cells/ml with or without 25 μg/ml OVA. Cultures were terminated andsupernatant fluid collected at 4 hours, and at 1, 3 and 5 days. Cytokineconcentrations were measured using an antigen-capture ELISA, based oncommercially available antibodies. Cytokine concentrations in culturesupernatants were determined using a standard curve and recombinantcytokines as the reference standard. The standard curve ranges were30-2000 pg/ml. ELISAs for the measurement of IL -2, IL -3, IL -4, IL -5,IL -6, IL-10 and granulocyte-macrophage-colony stimulating factor(GM-CSF) were performed. For gamma interferon, the standard curve rangewas 150-10,000 pg/ml.

Cytotoxic T-lymphocyte responses (CTL) were measured using a standard invitro assay and two different cells for stimulator and target cells. TheOVA.EG7 cell line was used as both target cells and antigen specificstimulator cells; cells were irradiated when used as stimulator cells.Control target cells were wild-type EL-4 cells, that are not transfectedwith OVA. Splenic mononuclear cells were used as the effector cells.These cells were assayed after in vitro stimulation to induce precursorCTL maturation to functional CTL effector cells.

C. Results

1. Measurement of OVA-Specific Antibody Responses

OVA-specific antibody responses were not induced 4 weeks following twoimmunizations with 15 μg of OVA in saline, as shown in FIG. 10. However,high-titered antibody responses were induced using complete Freund'sadjuvant, followed by incomplete Freund's adjuvant, and a W/Oformulation containing the CRL1005 copolymer. These data demonstrate thepoor immunogenicity of OVA when used at the selected dose withoutadjuvants and further demonstrate that this lack of immunogenicity canbe corrected using potent adjuvants.

Evaluation of alum, Ribi O/W adjuvant and two concentrations of theaqueous particulate CRL1005 demonstrated that these `clinicallyrelevant` adjuvants could partially correct the lack of immunogenicity(FIG. 11). The alum-based formulation induced significant antibodyresponses in 1/6 mice whereas the Ribi formulation induced responses in2/6 animals. The formulations containing 2.5% and 5.0% CRL1005 inducedsignificant antibody responses in 3/6 and 4/6 mice, respectively. TheQuil-A saponin adjuvant supplemented formulation did not inducesignificant antibody responses in any of the mice. These datademonstrate that the CRL1005 supplemented aqueous formulations are moreimmunogenic that those formulated with alum or Quil-A and are equal toor slightly better that the Ribi O/W formulation.

2. Measurement of Antigen-Induced In Vitro Cytokine production

Immunization with OVA alone did not induce measurable cytokineresponses, similar to the result observed for antibodies. The analysiswas then focused on the clinically relevant adjuvants, alum, saponin(Quil-A), Ribi O/W and CRL1005. None of these cytokines was detected inculture supernatants after only a 4 hour culture indicating that spleencells from immunized mice were not constitutively producing largeamounts of cytokines. Detectable levels were also not produced in vitroin response to antigenic stimulation with OVA at day 1 but weredetectable at days 3 and 5 (Table 2).

                  TABLE 2                                                         ______________________________________                                        Measurement of cytokine production by splenic mononuclear cells               from mice immunized with OVA and clinically relevant adjuvants                          ADJUVANT USED                                                       Cytokine                      Ribi  CRL1005                                   Detected (day)                                                                            Alum    Quil-A    (O/W) (2.5%)                                    ______________________________________                                        IL-2 (3)    --      --        --    35                                        IL-2 (5)    --      --        --    93                                        IL-3 (3)    --      --        --    --                                        IL-3 (5)    73      83        --    --                                        IL-4 (3)    --      --        --    --                                        IL-4 (5)    --      --        --    --                                        IL-5 (3)    781     42        --    101                                       IL-5 (5)    971     1720      38    1234                                      IL-6 (3)    --      --        38    --                                        IL-6 (5)    100     --        160   --                                        IL-10(5)    --      --        --    --                                        IL-10(5)    >2000   723       --    901                                       GM-CSF (3)  73      16        23    22                                        GM-CSF (5)  763     982       724   818                                       γ-IFN 95      316       --    371                                       IFNmma.     867     1749      880   1526                                      ______________________________________                                         Data are expressed as pg/ml. The standard curve ranges for all cytokines,     except γIFN, were 30-200 pg/ml. Data expressed as (--) represent        readings less than the lowest value on the standard curve.               

Cytokine production was readily detectable for groups of mice that didnot produce detectable levels of serum antibodies specific for OVA, suchas those immunized with Quil-A containing formulations. All of theadjuvants induced high levels of GM-CSF production whereas none appearedto induce IL-4 production and only low levels of IL-3 were produced.Only alum and Ribi adjuvants induced IL-6 products. Only those mice thatreceived formulations containing the CRL1005 copolymer produced highlevels of IL-2 and gamma interferon (γIFN) suggesting this adjuvantmight be a more potent inducer of Type 1 cytokine responses. The use ofhigher concentrations of CRL1005 did not significantly increase theproduction of any of these cytokines.

3. Measurement of OVA-Specific CTL Responses

Mice immunized with OVA in saline without an adjuvant were tested toestablish the background of the assay system. CTL activity against theEL-4 and EG-7.OVA target cells was not detected following culture ofspleen cells without antigen or with irradiated EG-7.OVA. These resultsindicate that CTL activity specific to OVA was not induced byimmunization with OVA in saline. Animals immunized with formulationscontaining the CRL1005 were tested in a similar manner. CTL specific forOVA were not detected following culture of spleen cells without antigen.However, CTL were readily detected after culture of spleen cells withthe EG-7.OVA cell line. The CTL killed only the EG-7.OVA cells, not theEL-4 cells, which demonstrated their antigen specificity. Since theEG-7.OVA cells express only class I major histocompatibility complex(MHC) antigens, the CTL activity is assumed to be the function ofCD8+T-lymphocytes. These results demonstrate that CTL activity specificto OVA was induced by vaccination with OVA formulated with CRL1005adjuvant in a simple aqueous solution.

The basis of the animal model was the selection of a dose of OVA thatwas non-immunogenic unless used with an adjuvant. Based on antibodyresponses, these studies demonstrated that the 15 μg/dose of OVA wasapparently non-immunogenic when used alone but was very immunogenic whenformulated with experimental W/O adjuvants. The adjuvants that are morelikely to be used in vaccines, the `clinically relevant` adjuvants, allaugmented antibody responses but to a lesser degree. The CRL1005 polymerwas more potent that saponin (Quil-A) and alum and at least equal inpotency to the Ribi O/W adjuvant.

While not wanting to be bound by this statement, it is believed, basedon the initial evaluation of cytokine production profiles, that theCRL1005 polymer induced both Type 1 and Type 2 responses since IL-2,γIFN, IL-5 and IL-10 were produced. Type 1 and 2 responses are definedbased on the cytokines produced by lymphocytes responding to an antigen.The Type 1 cytokine profile is the production of interleukin (IL) 2 andγIFN. The Type 2 profile is the production of IL-4, IL-5, IL-6 andIL-10.

EXAMPLE VIII

Rabbit Toxicology Trial and Stability Experiments

The CRL1005 polymer and related polymers are synthetic and manufacturedunder GMP standards. Thus they represent a more consistent product thatthose derived from natural sources such as bacterial endotoxins andsaponins. Rabbits have received three doses, administeredintramuscularly, and no toxic reactions have been observed.

CRL1005 has been, and is continuing to be, evaluated to determine itsstability. Currently, real-time stability data has been generated forboth the CRL1005 polymer in bulk (1 year) and in aqueous formulation (7months). Thus, this technology appears well suited for use in subunitvaccines.

EXAMPLE IX

Oral Delivery of Vaccines Containing Block Copolymer

The ability to deliver vaccine orally has two advantages over standardparenteral routes: (1) ease of administration and (2) the possibility ofinducing mucosal immune responses. The large copolymers are well suitedfor use in vaccine formulations because (1) they are nonionic andtherefore resistant to damage by stomach acids, (2) they inhibit lipaseactivity which should contribute to their utility with emulsionscontaining squalane, and (3) they can be used in aqueous formulationswhere they form appropriately sized particles.

The utility of the CRL1005 copolymer for use as an oral vaccineadjuvant/delivery system using OVA as the vaccine antigen was evaluated.To ensure immunogenicity of the formulation, C57BLJ6 mice in thepositive control group were immunized twice, at ten day intervals,subcutaneously (s.c.) with 25 μg/100 μl dose of OVA ±1.25% CRL1005 inPBS. Mice immunized orally received 125 μg/500 μl dose of ±1.25% CRL1005in bicarbonate buffer, again at 10 day intervals. Mice were alsoimmunized using nasal delivery since dilution or degradation in thestomach was considered to be a possibility. Mice received the sameamounts of OVA and CRL1005 used for the s c injections but in a volumeof 20 μl. Blood was collected as the source of serum antibodies fortesting 14 days after the second immunization.

The results of this study are shown in FIG. 13. Oral or nasalimmunization with OVA alone failed to induce antibody responsesdetectable using serum although s.c. immunization was effective. Theaddition of CRL1005 to the formulations increased the immunogenicity ofthe s.c. formulation and induced serum antibodies following both oraland nasal dose routes. These data demonstrate the utility of the CRL1005copolymer in vaccines to be delivered orally or nasally.

EXAMPLE X

This example demonstrates the effect of the certain of the copolymers ofthe present invention on gene transfer into mammalian cells.

Cells and reagents: Chinese hamster ovary cells (CHO-K1) and COS-7African green monkey kidney cells were obtained from ATCC, Rockville,Md. Cell culture media (Ham's F-12 medium, Dulbeccos Modified Eagle'sMedium), Hanks Balanced Salts, antibiotics (Penicillin-Streptomycin) andβ-galactosidase substrate X-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside) were obtained from Sigma,St. Louis, Mo. Fetal calf serum was obtained from Gibco-BRL, GrandIsland, N.Y. and Atlanta Biologicals, Atlanta, Ga. Lipofectin andOpti-MEM were obtained from Gibco BRL, Grand Island, N.Y. DOTAP wasobtained from Boehringer Mannheim, Indianapolis, Ind. β-galactosidaseplasmid pCMVβ (7.2 kb) was obtained from Clontech, Palo Alto, Calif.,pSVβ (6.74 kb) was obtained from Promega, Madison, Wis. pATCgD plasmidencoding HSV-1 glycoprotein D was obtained from Dr. Kousoulas, LSU,Baton Rouge, La.

Poloxamers: Non-ionic block copolymers (poloxamers) were synthesized atCytRx Corporation, Norcross, Ga. and stored in crystalline form at roomtemperature. Poloxamer compounds CRL-1012, CRL-1029, CRL-1190, CRL-1005,CRL-1023 and CRL-1030 were resuspended in sterile water at aconcentration of 10-20 mg/ml and stored in sealed bottles under nitrogenand at 4° C. These were diluted in tissue culture medium before addingthem to cell cultures.

In vitro Transfection with Liposomes: CHO and COS-7 cells were plated on24-well Costar plates, approximately 1×10⁵ in 1.0 ml of medium andincubated at 37° C. in a 5% CO₂ incubator. Cells were grown to 50%confluency and transfected with β-gal plasmids using as transfectionreagents either Lipofectin, DOTAP or poloxamers.

Transfection with Lipofectin was done as follows: Cell were washed twicewith low-serum medium (Opti-MEM), then the transfection mixture (200 μlof Opti-MEM at 37° C., 2 μl of Lipofectin and 1-2 μg of plasmid DNA,mixed in a polystyrene tube) was added and the cells were incubated for6 hrs at 37° C. in 5% CO₂. Following incubation, the transfectionmixture was removed by aspiration, fresh DMEM-10% FCS (0.8 ml) wasadded, and the cells were further incubated for 48 hrs. Transfectionswith DOTAP were done in a similar way with the difference thattransfection mixtures contained DMEM plus 10% FCS instead of low-serummedium Opti-MEM.

In vitro Transfection with Poloxamers: CytRx poloxamers CRL-1012,CRL-1029, CRL-1190, CRL-1005, CRL-1023 and CRL-1030 were screened fortheir ability to transfect DNA into mammalian cells in vitro using amethod based on the method described for liposome-mediated DNAtransfection. To optimize this method for efficient transfection thefollowing conditions were tested: mixing DNA (2 μg) with poloxamers at awide range of concentrations (5 μg/ml to 50 mg/ml); mixing DNA andpoloxamer in water, buffers (PBS, Tris, HBS), at various pH (5-7.5);addition of MgCl₂ (1-50 mM); mixing DNA and poloxamer at varioustemperatures (0-4° C., 25° C.-37° C. temperature shifts 4° C. to 37°C.); mixing by vortexing, sonication or syringe emulsification; presenceof serum in transfection mixtures; transfection of cells at differentconfluencies (20%, 50%), times (6 hr, 24 hr); and rotation of platesduring transfection.

After extensive studies to find the optimal conditions for in vitrotransfection with poloxamers, the following protocol was adopted. Thetransfection mixture was prepared in ice and consisted of 1 μl ofplasmid DNA (2 μg), 18 μl of poloxamer suspension in water (50-200μg/ml) for a total of 20 μl. The poloxamer-DNA mixture was incubated inice for 15 minutes, shifted to 37° C. and incubated for 5 minutes. After3 temperature shifts from 4° C. to 37° C., 180 μl of DMEM-10% FCS wereadded and the mixture (200 μl) was added to the cell monolayer at 50-60%confluency and the cells incubated for 24 hrs at 37° C. in 5% CO₂.Following incubation, the transfection mixture was removed byaspiration, fresh DMEM-10% FCS (1 ml) was added, and the cells werefurther incubated for 48 hrs.

X-Gal Staining of transfected cells: COS-7 and CHO cells transfectedwith β-galactosidase plasmids were tested after 48 hrs for expression ofβ-galactosidase by staining with the substrate X-Gal. The medium wasremoved and gently the cell monolayer was washed 2 times with PBS, pH7.3 (calcium and magnesium free). Cells were fixed for 30 min. at 37° C.in 1% formaldehyde in PBS (0.5 ml/well) and washed 2 times in PBS (1ml/well). Added 200 μl of X-Gal staining solution (800 μg/ml X-Gal indimethylformamide, 4 mM potassium ferrocyanide, 4 mM potassiumferricyanide, 2 mM MgCl₂ in PBS, pH 7.3) and incubated the cells for 24hrs at room temperature. After staining, the plates were washed 2 timesin PBS, 2% formaldehyde in PBS was added (1 ml/well) and observed underthe microscope for counting the number of cells stained dark blue.

Genetic Immunization and HSV ocular Infection: BALB/c 5-7 week oldfemale mice (4 per group) were immunized either i.p. or i.m. with pATCgDplasmid alone (20 μg or 50 μg), pATCgD (20 μg) plus DOTAP (50 μg or 100μg), and pATCgD (10 μg, 20 μg or 50 μg) plus CRL-1012 (50 μg, 200 μg or1 mg). Animals immunized i.p. received one inoculation of 200 μl;animals immunized i.m. received one inoculation (100 μl per leg) intoboth quadriceps. Animals received two inoculations every two weeks andserum samples were collected two weeks after each inoculation. Two weeksafter the last inoculation animals were infected with HSV-1 byscratching the corneal epithelium of one eye with a 25-gauge needle (6crisscross strokes) and by applying the virus suspension (1.6×10⁶pfu/ml) with a cotton-tipped applicator. Animals were examined under amicroscope 2-3 times a week for two weeks post-infection and once a weekthereafter. The eyes were graded on a scale 0 to +4 for blepharitis,stromal keratitis and vascularization. The results were expressed asseverity of disease, % morbidity and % mortality.

Genetic Immunization and HSV skin infection: SKH-1 hairless mice (5-7week old females) were immunized i.m. with pATCgD plasmid alone (20 μgor 50 μg), pATCgD (20 μg) plus DOTAP (50 μg or 100 μg), and pATCgD (20μg or 50 μg) plus CRL-1012 (50 μg or 200 μg). Each treatment groupincluded 4 mice. Animals received two inoculations animals (100 μl perleg) into both quadriceps every two weeks and serum samples werecollected two weeks after each inoculation. Two weeks after the lastinoculation animals were infected with HSV-1 by scratching the skin atthe base of the neck (1 cm) with a 25-gauge needle and by rubbing thescarified skin site with a cotton-tipped applicator dipped in a virussuspension (1.0×10¹¹ pfu/ml). Each group of animals were examined twotimes a week for two weeks and scored for size of the lesion (0 to 4), %morbidity and % mortality.

Antibody Screening by Indirect Immunofluorescence and ELISA: Sera fromanimals immunized with pATCgD plasmid alone or in combination withpoloxamers were tested for specific antibody response to HSV gD byindirect immunofluorescence (IIF) and ELISA. IIF was done with CHO cellstransiently expressing HSV gD at the cell surface after transfectionwith pATCgD and DOTAP (as described previously). Transfected cellmonolayers (in 8-well chamber slides) were incubated for 72 hrs to allowfor maximum cell surface expression of HSV gD. Cell monolayers werewashed twice in PBS, fixed in 1% formaldehyde in PBS and permeablized inmethanol at -20° C. for 3 minutes. Cells were washed in PBS andincubated in blocking buffer (1% FCS in PBS) for 30 minutes at roomtemperature. Cells were washed 3 times in PBS and incubated (1 hr atroom temp.) with sera from immunized animals diluted 1:100 in blockingbuffer. Cells were washed 3 times in PBS and incubated for 1 hr at roomtemp. in antibody conjugate (rabbit anti-mouse IgG FITC) diluted 1:1000in blocking buffer. Following labeling, the slides were washed in PBS,covered with a cover slip in mounting media and examined by fluorescencemicroscopy.

Antibody screening by ELISA was as follows: Vero cells cultured in Tflasks (75 cm²) were infected with HSV-1 (MOI of 5-10) and after 24 hrsharvested from the culture flasks and washed twice in PBS bycentrifugation. The pellet of infected cells was resuspended in 10 ml ofPBS and the cell suspension transferred to 96-well microtiter plates (50μl/well). The cell suspension was allowed to dry for 24 hrs, fixed withmethanol for 20 minutes and washed with PBS. Coated plates were storedat -20° C. until used. Sera from immunized animals were tested at 1:50to 1:1000 dilutions in PBS/0.05% Tween-20 (PBST), and the secondaryantibody consisted of goat anti-mouse IgG-HRP at 1:500 to 1:1000 inPBST. The color-producing substrate solution (40 mg o-phenylenediamine,0.05% H₂ O₂ in citrate phosphate buffer) was incubated for 30 minutesand the reaction was stopped by addition of 25 μl of 8N H₂ SO₄. Theabsorbance at 495 nm of each well was measured in a Molecular DevicesSpectraMax 250 microplate reader.

In vitro Transfection with Poloxamers: A series of poloxamer compoundswere tested, under different conditions, for in vitro transfection ofβ-galactosidase reporter genes (pCMVβ or pSVβ) into mammalian cells.Initial screening of poloxamers was done following a transfection methodbased on the method described for liposome-mediated DNA transfection.Under these conditions it was shown that poloxamers CRL-1012, 1023, 1029and 1030, at a concentration of 5 μg/ml, gave positive transfection,although the transfection efficiency was low (<1%) compared to thelevels of transfection typically obtained with the commercial reagentLipofectin (10-20%) in low serum medium (FIG. 14). Since DNAtransfection by Lipofectin is inhibited by serum in the culture medium,it was decided to test poloxamers in the presence of medium containing10% fetal bovine serum. Poloxamers 1012, 1029 and 1030 were shown tomediate DNA transfection at low levels (<1%) while transfection withLipofectin was strongly inhibited by serum (FIG. 15).

Further testing and screening of poloxamer compounds for in vitro DNAtransfer focused on compounds CRL-1012 and CRL-1029. A number ofconditions were tested in order to improve the efficiency oftransfection (see materials and methods). It was found that by mixingplasmid DNA (1-2 μg) and poloxamers (5-50 μg/ml) in ice (2° C.) andrepeated temperature shifts from 2° C. to 25° C., the transfectionefficiency was increased to levels of approximately 2-5% (FIG. 16).These poloxamers are water soluble at low temperatures presumably due tothe formation of hydrogen bonds between water molecules and ether-linkedoxygen groups in the POP block. At room temperature, or above the cloudpoint, poloxamers turn insoluble forming particulate aggregates.Although not wanting to be bound by the following theory, it is believedthat during this phase transition DNA molecules are trapped into theseaggregates which can be internalized by cells. In these studies, highvariability was observed in duplicate experiments and no correlationcould be established between poloxamer concentration and transfectionefficiency (FIG. 17).

It is possible that the formation of poloxamer-DNA aggregates is notvery efficient and that these aggregates are unstable due to thenon-ionic nature of poloxamers. It is also possible that under theconditions tested, poloxamer-DNA aggregates are not efficientlyinternalized by cells. It was found that mixing of poloxamer-DNAmixtures by vortexing or emulsification with a syringe, resulted in lossof DNA transfection activity (FIG. 18). Adding poloxamer-DNA mixtures tocells and rotating the plates for 24 hrs during the transfection wastested. Under those conditions, a slight enhancement of transfection wasobserved (FIG. 19). DNA transfections in larger volumes (6-well plates;400 μl per well) was tested to determine if by scaling-up the assayconditions, the efficiency of transfection could be increased. Theresults showed that although the reproducibility of the assay wasslightly improved, transfection efficiency was not increased (FIG. 20).

In summary, these results indicate that in vitro DNA transfection withpoloxamers CRL-1012 and CRL-1029, under the conditions tested, result intransfection efficiencies of 2-5%. In comparison to commercial reagents,poloxamers gave transfection levels higher than Lipofectin (>1%) in thepresence of serum, but lower than those obtained with DOTAP (20-30%) inserum. This is shown in the following Table.

Screening of CytRx Poloxamers for In Vitro Transfection

    ______________________________________                                        Poloxamer tested                                                                            Estimated % Transfection                                        ______________________________________                                        1005          neg                                                             1012          2-5%                                                            1023          <1%                                                             1027          neg                                                             1028          1-2%                                                            1029          2-5%                                                            1030          <1%                                                             1183          neg                                                             1190          <1%                                                             8131          neg                                                             Lipofection Controls                                                          Lipofectin    10-20%                                                          Lipofectin + FCS                                                                            <1%                                                             DOTAP + FCS   20-30%                                                          ______________________________________                                    

Further analysis is needed to define the optimal conditions andmechanism by which poloxamers mediate DNA uptake. It is possible thatpoloxamer-DNA complexes are taken-up by cellls or that poloxamersincrease cell membrane permeability allowing the uptake of DNA. Our datasupports the idea that poloxamers form aggregates entrapping DNA andthat these aggregates are internalized by cells. If poloxamer particlesare taken-up cells it would be of interest to develop these compounds asdelivery vehicles for many different applications. Development ofpoloxamers for DNA delivery may require modification of poloxamers byaddition of positively charged groups such as quaternary amino groups orconjugation to positively charged polylysine peptides. Positivelycharged poloxamers would bind DNA molecules through ionic interactionsresulting in the formation of more stable complexes for delivery tocells. In addition poloxamers linked to specific receptor ligands couldbe utilized for delivery of DNA to target specific organs and tissues byreceptor mediated endocytosis.

In vivo Gene Vaccination Studies: Antibody responses to HSV-1glycoprotein D: Initial studies tested the effect of poloxamer 1012 ingenetic vaccination with the plasmid pATCgD (pgD). BALB/c mice (4 pergroup) were immunized either i.p. or i.m. with pgD plasmid alone, incombination with liposomes (DOTAP) or in combination with poloxamer1012. After two inoculations every two weeks mice immunized with amixture of pgD plus 1012 at different concentrations (pgD 20 μg/1012 50μg, pgD 50 μg/1012 50 μg, and pgD 50 μg/1012 200 μg) showed positiveanti-gD responses by IIF. The best antibody responses were obtained inanimals inoculated intramuscularly (FIG. 21). The results obtained byIIF correlated with antibody titers obtained by ELISA. At serumdilutions of 1:200 and 1:400, the highest anti-gD responses were seen inanimals immunized with mixtures of pgD and 1012 (FIG. 21). These resultssuggest that 1012 increases the efficacy of gene vaccination with pgD,by enhancing the uptake of DNA and or by acting as an adjuvant topotentiate the immune response.

Genetic vaccination against ocular HSV-1 infection: BALB/c mice (4 pergroup) were inoculated i.m. with pgD plasmid alone, in combination withliposomes (DOTAP) or in combination with poloxamer CRL-1012. After twoinoculations every two weeks mice immunized with a mixture of pgD (50μg)/1012 (50 μg, 200 μg and 1 mg) and animals immunized with a mixtureof pgD (20 μg)/DOTAP (50 μg) showed higher anti-gD responses compared togroups immunized with pgD plasmid alone and mixtures of pgD/1012 atlower concentrations (FIG. 22).

The efficacy of genetic vaccination with pgD and 1012 was tested byinfecting animals through the corneal epithelium with HSV-1. Ocularinfection of naive mice resulted in high morbidity (88%) and mortality(77%) during 15 days post-infection. The lowest morbidity rate wasobserved in animals immunized with pgD alone (20-50 μg) and animalsimmunized with pgD (20 μg) plus 1012 (200 μg, 1 mg). No mortality wasobserved in animals immunized with pgD (20 μg)/DOTAP (50 μg) and animalsimmunized with pgD (50 μg)/1012 (1 mg). Groups of mice immunized withpgD (50 μg)/1012 (50, 200 or 1 mg) showed high morbidity but only onemouse died (25%) of HSV infection (FIG. 23). In these experiments apositive correlation was observed between anti-gD antibody titersobtained by ELISA and % morbidity and mortality after a challengeinfection with HSV-1.

Genetic vaccination against skin HSV-1 infection in hairless mice: SKH-1hairless mice immunized i.m. with pgD plasmid alone, in combination withDOTAP, or in combination with poloxamer 1012, and tested for immunityagainst a skin infection with HSV-1. Infection of naive mice resulted in100% morbidity and 50% mortality during the first 15 dayspost-infection. Animals immunized with pgD alone (20 or 50 μg) showedhigh morbidity and no mortality. Animals immunized with pgD 20 μg/1012200 μg and pgD 50 μg/1012 50μg showed 100% morbidity but only one mousedied (25%) from HSV infection. Animals immunized with pgD (50 μg)/1012200 μg showed 80% morbidity but no mortality. The worse disease observedwas in animals immunized with pgD (50 μg) plus DOTAP (100 μg), showing100% mortality. Only in this group of animals systemic HSV disease wasobserved (FIG. 24). The severity of the disease was also assessed byscoring the size of the lesions (0 to 4). Less severe lesions, such asredness of the skin at the site of infection, were seen in animalsimmunized with pgD alone (50 μg) and animals immunized with pgD (50μg)/1012 (50 μg) (FIG. 25).

It should be understood, of course, that the foregoing relates only topreferred embodiments of the present invention and that numerousmodifications or alterations may be made therein without departing fromthe spirit and the scope of the invention as set forth in the appendedclaims.

What is claimed is:
 1. A method of synthesizing a composition consistingof a polyoxypropylene/polyoxyethylene block copolymer having thefollowing general formula:

    HO(C.sub.2 H.sub.4 O).sub.a (C.sub.3 H.sub.6 O).sub.b (C.sub.2 H.sub.4 O).sub.a H

wherein "b" represents a number such that the molecular weight of thehydrophobe (C₃ H₆ O) is greater than 15,000 Daltons and "a" represents anumber such that the percentage of hydrophile (C₂ H₄ O) is betweenapproximately 3% and 30% by weight, said method comprising: (a) addingpropylene glycol to an oxygen free reactor vessel containing cesiumhydroxide (CsOH.H₂ O) and maintaining a temperature of betweenapproximately 90° C. and 120° C., wherein the mole ratio of the cesiumhydroxide to the propylene glycol is between approximately 12 to 1:8;(b) adding propylene oxide to the reactor vessel under conditionsallowing the formation of a polyoxypropylene molecule, the propyleneoxide being added by rate limiting vapor phase addition, minimizingexcess propylene oxide, and the propylene oxide being added until themolecular weight of the polyoxypropylene molecule is greater than 15,000Daltons; (c) adding ethylene oxide to the reactor vessel underconditions allowing the formation of polyoxyethylene blocks on each endof the polyoxypropylene molecule, thereby formingpolyoxypropylene/polyoxyethylene block copolymer in the reactor vessel,the ethylene oxide being added until the percentage of polyoxyethyleneis between approximately 3% to 30% by weight of the block copolymer, theblock copolymer having significantly reduced level of unsaturation. (d)removing any remaining cesium hydroxide in the block copolymer.
 2. Themethod of claim 1, wherein "b" represents a number such that themolecular weight of the hydrophobe (C₃ H₆ O) is between greater than15,000 Daltons and 20,000 Daltons.
 3. The method of claim 1, wherein "a"represents a number such that the percentage of hydrophile (C₂ H₄ O) isbetween approximately 5% and 30% by weight.
 4. The method of claim 1,wherein "a" represents a number such that the percentage of hydrophile(C₂ H₄ O) is between approximately 5% and 25% by weight.
 5. The methodof claim 1, wherein in b), the temperature is approximately 100° C.